1. Field of the Invention
Agents that alter the transport activity of small, neutrally charged solutes by solute transporters are needed as therapeutic agents for increasing solute clearance in states of fluid overload and for treating diseases and conditions such as hypertension. Methods for identifying and using agents that inhibit solute transporters such as urea transporters are described herein.
2. Description of the Related Art
Urea is generated as the major end product of hepatic nitrogen metabolism and is excreted primarily by the kidney. Urea and sodium chloride are the major solutes in the hyperosmolar renal medulla. In the antidiuretic kidney, urea is greatly concentrated with respect to plasma (up to 100 times in humans and 250 times in rodents) by countercurrent multiplication and exchange mechanisms (Bankir et al., In The Kidney (6th Edition), pages 637-679, Brenner, B M, ed., (WB Saunders Company, Philadelphia, Pa.) (2000)). Of central importance to these mechanisms is intrarenal urea recycling, which requires facilitated urea transport by molecular urea transporters (UTs). UTs are comprised of two major subfamilies encoded by different genes (UT-A and UT-B) (see, e.g., Bagnasco, Am. J. Physiol. 284:F3-F10 (2003); Shayakul et al., Pflügers Arch. 447:603-609 (2004); Yang et al., J. Biol. Chem. 273:9369-72 (1998)). In kidney, a single UT-B isoform is expressed in vasa recta while several splice variant UT-A-type transporters are expressed in kidney tubule epithelia (see, e.g., Sands, Curr. Opin. Nephrol. Hypertens. 13:525-32 (2004)).
Phenotype analysis of mice separately lacking vasa recta UT-B or inner medullary collecting duct UT-A1/3 implicated UT involvement in the formation of concentrated urine and in renal urea clearance (see, e.g., Yang et al., J. Biol. Chem. 277:10633-37 (2002); Fenton et al., Proc. Natl. Acad. Sci. USA 101:7469-74 (2004); Fenton et al., J. Am. Soc. Nephrol. 16, 1583-92 (2005)). The UT-B knock-out mice that were generated manifested a urea-selective urinary concentrating defect associated with urinary hypoosmolality and increased renal urea clearance (Yang et al., supra). UT-B is also expressed outside of the kidney, most notably and in highest abundance in red blood cell (RBC) membranes. Loss-of-function human UT-B mutations result in greatly reduced urea permeability in RBC and a mild urinary concentrating defect (Sands et al., J. Am. Soc. Nephrol. 2:1689-96 (1992); Lucien et al., J. Biol. Chem. 273:12973-80 (1998)).
Diuretics are administered widely in humans to increase renal salt and water clearance in a variety of conditions that are associated with total body fluid overload, such as congestive heart failure and cirrhosis, as well in normovolemic states such as hypertension and syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Most diuretics are inhibitors of salt absorption by kidney tubules, such as a furosemide block of Na+/K+/2Cl− co-transport in the thick ascending limb of Henle and a thiazide block of Na+/Cl− co-transport in the distal tubule. Recently, a new type of diuretic, called an “aquaretic,” has been developed to increase renal water clearance in hyponatremia associated with fluid overload or SIADH (see, e.g., Goldsmith, Am. J. Cardiol. 95:14B-23B (2005); Miller, J. Am. Geriatr. Soc. 54:345-53 (2006)). Vasopressin-2 receptor (V2R) antagonist aquaretics have been approved for clinical use in some countries, though not yet in the United States, and aquaporin inhibitor aquaretics are under development.
Functional studies in knock-out mice indicate a critical role for urea transporters (UTs) in the urinary concentrating mechanism and in renal urea clearance. However, potent and specific urea transport blockers have not been available. Accordingly, a third type of diuretic is needed: one that targets renal urea clearance mechanisms. Because urea is of at least equal importance to NaCl in the renal countercurrent mechanism for urinary concentration (see, e.g., Bankir et al., supra; Masilamani et al., In The Kidney (6th Edition), Brenner, ed. Philadelphia, Pa.; WB Saunders Company; pages 595-35; (2000)), such diuretics are needed for increasing solute clearance in states of fluid overload, hypertension, and may also be useful in prolonging dialysis-free survival in chronic renal insufficiency. A need also exists for methods for rapid screening of compounds to identify potential urea transporter inhibitors and other compounds that effectively increase renal water and solute clearance for subjects who are exhibiting a water-retaining state.